The majority of phosphorus (80-85%) present in the body is found in the bones as hydroxyapatite. The remainder is present as inorganic phosphate and phosphate esters. A determination of the concentration of inorganic phosphate in various body fluids can be highly useful in the diagnosis of various disease states. For example, increased serum phosphorus can be indicative of hypervitaminosis D. hypoparathyroidism, and renal failure. Similarly, reduced serum phosphorus levels are seen in rickets (Vitamin D deficiency), hyperparathyroidism, and Fancony's syndrome.
The inorganic phosphate content of body fluids has typically been determined using one of two known methods, or variations thereof. In the first method, that of Fiske and Subbarow [Biol. Chem. 66, 375 (1925)], a heteropoly compound formed between ammonium molybdate and inorganic phosphate is reduced under mild conditions to form the phosphomolybdenum complex, which is measured photometrically. In the second more widely used method, that of Daly and Ertingshausen [Clin. Chem. 18, 263 (1972)], inorganic phosphate is reacted with ammonium molybdate in sulfuric acid solution to form the unreduced phosphomolybdate complex, which is measured photometrically at about 340 nm.
Due to the immense volume of testing done by modern clinical laboratories, as well as the need for consistently accurate test results, inorganic phosphate determinations are typically performed using high speed, automatic instrumentation. In particular, the use of so-called centrifugal analyzers for the determination of inorganic phosphate has become widespread. An exemplary device of this type is described in U.S. Pat. No. 3,555,284.
Generally speaking, centrifugal photometric analyzers comprise an array of transparent reaction chambers or cuvettes arranged around the periphery of a centrifuge rotor. One or more solution receiving chambers are located radially inwardly from each cuvette and passageways are provided communicating between each cuvette and its associated solution receiving chambers. The solution receiving chambers are shaped to retain liquid when the rotor is at rest and to release liquids to the cuvettes when the rotor is spun. Thus, spinning of the rotor causes the transfer of sample, reagent or both to the cuvettes, where the samples and reagents react. As the rotor continues to spin, each cuvette passes, in turn, between a light source and a photodetector where the phototransmittance of the contents of each cuvette is read. These readings are then used to determine the concentration of a given species in each of the samples.
A typical prior art approach for the determination of inorganic phosphate utilizing a centrifugal analyzer is described in U.S. Pat. No. 3,795,484. In this method, which makes use of the unreduced phosphomolybdate complex chemistry described previously, samples under study are reacted in their respective cuvettes with a single reagent, which comprises ammonium molybdate, sulfuric acid and surfactant. Approximately two seconds after the rotor has begun to spin and, presumably, before the reaction has proceeded to a significant degree, a first or "blank" absorbance reading is taken for each cuvette. The purpose of this reading is to determine the inherent absorbance of the cuvette and unreacted sample and reagent. A second reading is taken after 10 minutes, the time required for the adequate reaction of sample and reagent. The change in absorbance between the first and second readings is compared to the change in absorbance obtained in a like manner for a standard having a known concentration of inorganic phosphate, in order to determine the phosphate content of each sample.
In an improvement of the above method, described in U.S. Pat. No. 4,220,451, a reduction of the time required for adequate reaction of sample and reagent--from 10 to about 2 to 4 minutes--is accomplished by optimizing the surfactant concentration of the reagent.
Although the above mentioned analytical methods certainly represent significant advances in the field of clinical chemistry, it will be appreciated that they nevertheless possess certain deficiencies. For example, in these prior methods the initial absorbance readings are not true blank readings, in that they are made after the reaction has already proceeded to an extent. Further, in view of the heavy workload of many clinical laboratories, the time required in these methods for reaction to proceed to an adequate extent and for a final reading to be taken, even at 2 to 4 minutes, is too long.
Accordingly, it is the object of this invention to provide an improved method for the determination of inorganic phosphate in fluids.
More particular objects are to provide an improved method for determining inorganic phosphate concentration which can be conveniently carried out using a centrifugal photometric analyzer, which allows for true sample blanking, and which is significantly more rapid than known methods.